Quinolone-Hydroxyquinoline Tautomerism in Quinolone 3-Esters. Preserving the 4-Oxoquinoline Structure To Retain Antimalarial Activity

Article abstract of DOI:10.1021/acs.joc.5b02169

Recent publications report in vitro activity of quinolone 3-esters against the bc₁ protein complex of Plasmodium falciparum and the parasite. Docking studies performed in silico at the yeast Q_o site established a key role for the 4-o

Full text of DOI:10.1021/acs.joc.5b02169

Article
pubs.acs.org/joc
Quinolone−Hydroxyquinoline Tautomerism in Quinolone 3‑Esters.
Preserving the 4‑Oxoquinoline Structure To Retain Antimalarial
Activity
Pedro Horta,†,‡ Nihal Kus,§,∥ Marta Sofia C. Henriques,⊥ Jose A. Paixao,⊥ Lis Coelho,# Fatima Nogueira,#
́ ́
̧
̃
Paul M. O’Neill,‡ Rui Fausto,*,§ and Maria Lurdes Santos Cristiano*,†
†CCMAR and Department of Chemistry and Pharmacy, FCT, University of Algarve, P-8005-039 Faro, Portugal
‡Department of Chemistry, University of Liverpool, Liverpool L69 7ZD, United Kingdom
§CQC, Department of Chemistry, University of Coimbra, P-3004-535 Coimbra, Portugal
∥Department of Physics, Anadolu University, 26470 Eskise
̧ hir, Turkey
⊥CFisUC, Department of Physics, University of Coimbra, P-3004-516 Coimbra, Portugal
#CMDT and Institute of Hygiene and Tropical Medicine, New University of Lisbon, P-1349-008 Lisboa, Portugal
S
* Supporting Information
ABSTRACT: Recent publications report in vitro activity of
quinolone 3-esters against the bc1 protein complex of
Plasmodium falciparum and the parasite. Docking studies
performed in silico at the yeast Qo site established a key role
for the 4-oxo and N−H groups in drug-target interactions.
Thus, the possibility of 4-oxoquinoline/4-hydroxyquinoline
tautomerism may impact in pharmacologic profiles and should
be investigated. We describe the synthesis, structure, photo-
chemistry, and activity against multidrug-resistant P. falciparum
strain Dd2 of ethyl 4-oxo-7-methylquinoline-3-carboxylate
(7Me-OQE) and ethyl 4-hydroxy-5-methylquinoline-3-carbox-
ylate (5Me-HQE), obtained from diethyl 2-[((3-methylphenyl)amino)methylene]malonate. Theoretically (B3LYP/6-311+
+G(d,p)), 5Me-HQE and 7Me-OQE show clear preference for the hydroxyquinoline form. The difference between the lowest
energy hydroxyquinoline and quinolone forms is 27 and 38 kJ mol−1, for 5Me-HQE and 7Me-OQE, respectively. Calculations of
aromaticity indexes show that in 5Me-HQE both rings are aromatic, while in the corresponding oxo tautomers the nitrogen-
containing ring is essentially non-aromatic. The structure of monomeric 5Me-HQE was studied using matrix isolation coupled to
FTIR spectroscopy. No traces of 4-oxoquinoline tautomers were found in the experimental IR spectra, revealing that the species
present in the crystal, 5Me-HQE·HCl, was lost HCl upon sublimation but did not tautomerize. Continuous broadband
irradiation (λ > 220 nm; 130 min) of the matrix led to only partial photodecomposition of 5Me-HQE (ca. 1/3).
with improved pharmacological properties, targeting a variety of
applications, e.g., cancer,4 hepatitis B,5 hepatitis C,6 HIV,7
herpes,8 fungal infections, immunomodulation,9 tuberculosis,10
or malaria.11−14
INTRODUCTION
■
The use of quinolones as antibacterial agents began in 1963.
Following the discovery of nalidixic acid, a naphthyridone 3-
acid, several quinolone derivatives with improved pharmaco-
logic profile were synthesized. Among these, the fluoroquino-
lones norfloxacin, ciprofloxacin, and levofloxacin (structures are
presented in Figure S1, Supporting Information) emerged as
the most efficient quinolone-type antibacterial drugs and are
widely used as second line antibiotics.1,2 As shown (Figure S1),
these quinolone-based antibacterial agents are N-alkyl-4-
oxoquinoline 3-acids. The N-alkyl substituent was introduced
to adjust pharmacologic properties.3 Also, N-substitution
hinders the possibility of quinolone-hydroxyquinoline tauto-
merism, restricting structural diversity of the active pharma-
ceutical ingredient.
Malaria remains one of the most deadly infectious diseases.
The growing spread of resistance by Plasmodium falciparum
against conventional antimalarial drugs boosted the search for
new chemotherapeutic solutions.15 The approval of malarone
for the treatment and prevention of multidrug-resistant malaria
validated the P. falciparum bc1 protein complex as target for
developing new antiplasmodial drugs. The bc1 complex is a
homodimeric transmembrane protein responsible for the
transfer of electrons from ubiquinol to cytochrome c, together
with the vectorial translocation of protons across the inner
The versatility of the quinolone chemotype has attracted
Received: September 15, 2015
intense research, yielding quinolones (mainly 4-oxoquinolines)
© XXXX American Chemical Society
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mitochondrial membrane. Loss of bc1 activity leads to loss of
mitochondrial function (relevant to provide intermediates for
pyrimidine and ATP synthesis) as evidenced by the collapse of
the transmembrane electrochemical potential, resulting in
parasite death.16,17 Figure 1 presents the structures of
Figure 2. 4-Oxo/4-hydroxy tautomeric equilibrium in 7-substituted
quinoline 3-esters.
Thus, we envisaged investigating the tautomer and con-
former preferences in quinolone 3-esters, using ethyl 4-oxo-7-
methylquinoline-3-carboxylate (Figure 2; 5; R = CH3) as our
model compound. For simplicity, we refer to this compound as
7-methyl-oxoquinoline-ester (7Me-OQE).
We describe herein the synthesis and structure of the
quinolone 3-ester 7Me-OQE. This compound was obtained by
thermal cyclization of diethyl 2-[((3-methylphenyl)amino)
methylene]malonate 9 in dowtherm A at 250 °C (see Scheme
1, route c) and isolated as sole product. The corresponding 4-
hydroxyquinoline tautomer (Figure 2; 6; R = CH3) was not
observed. Surprisingly, cyclization of compound 9 mediated by
POCl3, at 100 °C, led to a mixture of an amorphous powder
and crystals. Analysis of one of these crystals by X-ray
crystallography revealed the presence of the hydrochloride
salt of one hydroxyquinoline 3-ester isomer, ethyl 4-hydroxy-5-
methylquinoline-3-carboxylate 10 (see Scheme 1, route d; HCl
is formed as byproduct from POCl3-mediated cyclization). For
simplicity, we refer to 4-hydroxyquinoline 10 as 5-methyl-
hydroxyquinoline-ester (5Me-HQE) and to its hydrochloride
salt as 5Me-HQE·HCl. The crystal structure of 5Me-HQE·HCl
is described. The structure of monomeric 5Me-HQE was
investigated, using matrix isolation coupled to FTIR spectros-
copy (upon sublimation of 5Me-HQE·HCl, HCl is evolved and
sole the free base, 5Me-HQE, is isolated in the argon matrix)
and theoretical calculations (B3LYP/6-311++G(d,p)). A de-
tailed analysis of conformational and tautomeric preferences for
5Me-HQE is provided, and the possibility of quinolone-
hydroxyquinoline equilibria is evaluated. The conformational
and tautomeric preferences for monomeric 4-oxoquinoline
7Me-OQE were also theoretically investigated, at the same
level of theory.
Figure 1. Structure of quinolones with relevant antiplasmodial activity.
representative quinolone-type compounds with relevant anti-
plasmodial activity, targeting the bc1 protein complex of P.
falciparum. Endochin (1)18 was validated as one of the first
synthetic antimalarials. It exhibits activity against avian malaria
and served as model for new endochin analogues (endochin-
like quinolones, ELQ) with improved therapeutic properties.19
From these, the quinolone 3-diarylether ELQ-300 (2), an
antimalarial preclinical candidate,20 and the quinolone 3-ester
ICI 56,780 (3)21 emerged as leads.
In recent years, a range of 6- and 7-substituted quinolone 3-
esters have been prepared and tested in vitro against P.
falciparum malaria parasites. Interestingly, some 7-substituted
derivatives (4) expressed activity at low nanomolar concen-
trations.14 Docking studies performed in silico at the yeast Qo
site of the bc1 protein complex of P. falciparum suggested a key
role for residues His182 and Glu272 (in the target ISP) in the
recognition of high-potency inhibitors. The same studies also
indicated that the 4-oxoquinoline moiety is oriented so as to
enable formation of an H-bond between the quinolone
carbonyl and a protonated imidazole N atom of His181,
while a second H-bond (water mediated) is predicted between
the carboxyl group of the cytochrome b residue Glu272 and the
quinolone N−H. Thus, both the 4-oxo substituent and the N−
H group appear to be important to the inhibitory activity of
quinolone 3-esters toward P. falciparum bc1 protein complex.14
It is noteworthy that, unlike for the antibacterial fluoroquino-
lone drugs (see Figure S1), compounds 1−4 (Figure 1) bear
these structural characteristics.
Although some 7-substituted quinolone 3-esters appear
highly promising as drug leads, the compounds present
liabilities, such as poor aqueous solubility and low oral
bioavailability. Preliminary results from structure−activity
relationship (SAR) studies indicated that optimization could
be achieved by tuning the nature of substituent at position 7.14
Additionally, considering that the information from docking
studies in silico establishes a relevant role for the 4-oxo and N−
H groups in drug−target interactions, the possibility of
tautomerism between 4-oxoquinoline and 4-hydroxyquinoline
forms (5, 6; Figure 2) should not be neglected. Tautomeriza-
tion will translate in alteration of chemical and physical
properties, with possible implications in pharmacokinetic and
pharmacodynamic profiles.
The photostability of 5Me-HQE was assessed through an
investigation of its UV-induced matrix photochemistry. The
antiplasmodial activity of 4-oxoquinoline 7Me-OQE and 4-
hydroxyquinoline 5Me-HQE against the chloroquine and
mefloquine-resistant P. falciparum strain Dd222 was evaluated.
RESULTS AND DISCUSSION
■
Synthesis. Quinolone 3-esters may be prepared from an
α,β-unsaturated ester derivative of aniline (usually a malonate
derivative) through a thermally driven intramolecular cycliza-
tion. This approach, known as the Gould−Jacobs method-
ology,23 presents limitations, mostly related to the high
temperature (above 225 °C) required for reaction. Cyclization
was found to be concentration dependent, and the product may
undergo thermal degradation.8 Because most quinolones
exhibit low solubility, leading to difficulties in extraction and
purification, an alternative methodology for cyclization, using
phosphoryl chloride, was proposed. This approach involves
cyclization of the enamine and chlorination at the 4-position, in
one pot.24 It is anticipated that the 4-chloroquinoline produced
will be more easily isolated and may be subsequently converted
into the final quinolone. Also, the 4-chroroquinoline may be
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Scheme 1. Synthetic Approach to Ethyl 4-oxo-7-Methylquinoline-3-carboxylate, 7Me-OQE (5, R = CH3), and to Ethyl 4-
a
Hydroxy-5-methylquinoline-3-carboxylate, 5Me-HQE (10)
a
Conditions: (a) NH4Cl, Fe(II), MeOH:H2O (1:1), 60 °C, 4 h; (b) 100 °C, o.n.; (c) Dowtherm A, 250 °C, 3 h; (d) POCl3, 97 °C, o.n.
Figure 3. Crystal structure of ethyl 4-hydroxy-5-methyl quinolinium chloride 3-carboxylate (chloride salt of 10), showing (a) the intramolecular H-
bonding pattern; (b) molecular stacking and interactions between π electron clouds of aromatic rings. The ethyl group is disordered over two
alternate positions; for clarity, only the atoms of the position of larger occupancy are shown in the figure. An ORTEPII plot of the 5Me-HQE·HCl
molecule is provided in Figure S11.
C
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used as an intermediate for the synthesis of quinoline
derivatives.25
ppm), compared to the corresponding resonances in d6-DMSO
(H2, Ar−H, d, δ = 8.38 ppm; H8, Ar−H, s, δ = 7.44 ppm).
This difference is ascribed to the dissociation of the salt in d6-
DMSO, with loss of HCl and release of the free base. The H
NMR spectrum of 7Me-OQE was also measured in d-
The synthetic approach that led to quinolone 7Me-OQE and
of hydroxyquinoline 5Me-HQE (or corresponding salt, 5Me-
HQE·HCl) was performed as depicted in Scheme 1. Both
compounds were prepared from 3-nitrotoluene 7, in three
steps. 3-Nitrotoluene 7 was initially reduced to 3-methylaniline
8, which was converted into the diethylmalonate derivative 9.
Both steps (a, b; Scheme 1) were optimized and afforded high
yields of the isolated products.
The crystal structure of compound 9 was determined by X-
ray crystallography (Figure S4). Atomic coordinates, bond
lengths, valence and dihedral angles, and other crystallographic
data were deposited at the Cambridge Crystallographic Data
Centre, with the reference CCDC 1039617 (see Figure S4 for
crystal structure and interpretation of crystallographic data).
Bond lengths and valence angles of the diethyl 2-[((3-
methylphenyl)amino) methylene]malonate molecule 9 are
within the expected values.
1
chloroform (see Figure S7). For this compound, there was
1
no evidence in the spectra of significant differences in the H-
chemical shifts in d6-DMSO and in d-chloroform. So, it appears
reasonable to deduce that the differences in chemical shifts
observed for 5Me-HQE in both solvents were due to loss of
HCl from the salt with formation of the free base, induced by
the more polar media.
The 13C NMR spectrum of the crystalline 5Me-HQE in d6-
DMSO (see Figure S9) shows characteristic signals for
compound 10, corresponding to resonances of C4 (see Scheme
1 for atom numbering), at δ = 171.1 ppm (predicted at δ =
184.2 ppm for the quinolone tautomer), C8 and C8a, at δ =
127.9 and 161.3 ppm (predicted at δ = 114.5 and 141.4 ppm,
respectively, for the quinolone tautomer), demonstrating the
sole presence of the 4-hydroxyquinoline tautomer (5Me-HQE)
in solution. Figures S8 and S9 present the chemical shifts
As shown in Scheme 1, two methods were followed for the
preparation of quinoline derivatives from 9. Thermal cyclization
of 9 in Dowtherm A at 250 °C (Gould−Jacobs methodology)
afforded quinolone 7Me-OQE (5; R = CH3, route c). However,
cyclization of 9 mediated by POCl3, at 100 °C, led to formation
of hydroxyquinoline 5Me-HQE (10; route d). According to the
literature24 this strategy should afford 4-chloroquinoline, but, in
our case, a mixture of hydroxyquinoline (10, 5Me-HQE) and
its hydrochloride salt (5Me-HQE·HCl) was recovered.
In the thermally driven cyclization it is proposed that the
phenyl ring acts as nucleophile, attacking the carbon of the
ester carbonyl group.23 It is worth noticing that routes c and d
afforded quinolines with different patterns of aromatic
substitution, resulting from cyclization on both ortho carbons
of the aniline derivative 9.
1
(ppm) predicted for H- and 13C NMR spectra of 5Me-HQE
and the corresponding experimental spectra, in d6-DMSO.The
data obtained from elemental analysis of the powder obtained
from step d (Scheme 1) are compatible to calculated values for
the free base, 5Me-HQE.
Crystal Structure of 5Me-HQE·HCl. The crystal structure
of the hydrochloride salt of 5Me-HQE is presented in Figure 3.
Atomic coordinates, bond lengths, valence angles, dihedral
angles, and other crystallographic data were deposited at the
Cambridge Crystallographic Data Centre, with the reference
CCDC 1039599.
Each unit cell contains two anion−cation pairs, related by an
inversion center. Bond lengths and valence angles are within
the expected values. Two strong H-bonds are evidenced, one
between the amine group and the halide ion (N1···Cl: 2.955(2)
Å), the other between the hydroxyl substituent at C4 and the
oxygen of the ester carbonyl group (O15···O18:2.570 (3) Å).
In addition, a short contact distance C−H···Cl of 3.645(4) Å
involving the C6 atom can be spotted in the structure. The
major intermolecular interactions stabilizing the crystal
structure are probably weak dispersion forces (π−π type
interactions) between the π electron clouds of the aromatic
carbocyclic rings. The stacking of the molecules is such that the
electron clouds of each N-substituted ring in one layer interact
with those of two methyl-substituted phenyl rings in adjacent
layers and vice versa, as depicted in Figure 3b. The distances
between the centers of gravity of the rings (Cg1: N1−C2−C3−
C4−C9−C10; Cg2: C5−C6−C7−C8−C10−C9) in these four
intermolecular interactions are typical for parallel interaction
between two aromatic rings (Cg1−Cg2i: 3.629(2); Cg1−Cg2ii:
3.625(2); Cg1−Cg2i: 3.630(2); Cg1−Cg2ii: 3.624(2); i = −x,1
− y,1 − z; ii = 1 − x,1 − y,1 − z).
ATR-FTIR Experiments. 7Me-OQE and 5Me-HQE may
also be distinguished by FTIR spectroscopy. The ATR spectra
of crystalline samples of the two compounds and the proposed
band assignments are shown in Figure S12 and Tables S2 and
S3. As it could have been anticipated, the two spectra do not
differ much, except in the characteristic regions of the OH
group, present in 5Me-HQE and absent in 7Me-OQE, and for
the carbonyl stretching mode of the quinolone carbonyl group
of this latter compound, absent in the first. Specifically, (i) the
spectrum of 5Me-HQE is by far more complex in the high-
1
7Me-OQE and 5Me-HQE are easily distinguished by H
NMR and 13C NMR spectroscopy (see Scheme 1 for atom
numbering in 4-oxoquinoline and 4-hydroxyquinoline struc-
tures). Important 1H NMR signals (in d6-DMSO) for
identification of quinolone 7Me-OQE are those due to
resonances of H1 (N−H; bs, δ = 12.17 ppm) and H8 (Ar−
H; s, δ = 7.38 ppm). Relevant 13C NMR signals (in d6-DMSO)
for 7Me-OQE are those due to resonance of the carbonyl
carbon of the oxoquinoline system, C4, and to the carbonyl
carbon of the ester group, C10 (C4 δ = 176.0 ppm; C10 δ =
165.0 ppm). These signals confirm formation of 4-oxoquinoline
7Me-OQE from step c (Scheme 1). Figures S5 and S6 present
1
predicted H NMR and 13C NMR chemical shifts (ppm) and
the corresponding experimental 1H NMR and 13C NMR
spectra for 7Me-OQE in d6-DMSO.
Analysis of the product isolated from step d (Scheme 1)
revealed the presence of 4-hydroxyquinoline 5Me-HQE (10,
Scheme 1). In this case, cyclization led to a 4-hydroxyquinoline
3-ester methyl-substituted at position 5.
1
The H NMR spectra of crystalline hydroxyquinoline 5Me-
HQE (see Figures S8 and S10), in d6-DMSO and in d-
chloroform, show the absence of a signal corresponding to the
resonance of the proton from the N−H bond. Despite
presenting coincident splitting patterns that are compatible
1
with the hydroxyquinoline form, the H-chemical shifts are
different in d6-DMSO and in d-chloroform, the difference being
more pronounced for signals corresponding to H2 and H8. The
proton resonances for 5Me-HQE in d-chloroform appear
downfield (H2, Ar−H, d, δ = 9.26 ppm; H8, Ar−H, s, δ = 8.51
D
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frequency region, where bands due to the νOH mode are
observed (this profile is complicated by the coupling of this
mode with low-frequency intermolecular modes and involve-
ment of the OH group in the intramolecular H-bond with the
ester group), (ii) a band is observed at 1426 cm−1 in the
spectrum of 5Me-HQE, which has no counterpart in the
spectrum of 7Me-OQE, ascribable to the δCOH bending, (iii)
a shoulder due to the stretching mode of the quinolone
carbonyl group of 7Me-OQE is observed at 1724 cm−1, at a
position where no absorption is observed in the spectrum of
5Me-HQE. The τOH mode of 5Me-HQE was tentatively
assigned to the band at 861 cm−1 in the spectrum of 5Me-
HQE, but the complexity of the two spectra in this frequency
range precludes a definitive assignment of this vibration. On the
other hand, since the spectrum of 5Me-HQE corresponds to
that of the HCl salt of the compound, the quinolinic N is
protonated in both compounds, and the vibrations of this
moiety are observed at similar frequencies (see Tables S2 and
S3).
To assess the relative importance of the different tautomers
of the compound as isolated species (or in the gas phase), we
investigated the structure of monomeric 5Me-HQE, using the
matrix isolation technique coupled to FTIR spectroscopy and
theoretical methods (DFT/B3LYP/6-311++G(d,p)). The
7Me-substituted analogue was also investigated theoretically,
at the same level of theory, for comparison.
Theoretical Calculations. Investigation of the potential
energy surface of the 4-hydroxyquinoline tautomer 5Me-HQE,
at the B3LYP/6-311++G(d,p) level of computation, yielded 8
nonequivalent-by-symmetry conformers, graphically depicted in
Figure 5. Relative energies of these conformers are given in
The crystalline 5Me-HQE·HCl was washed with an aqueous
solution of sodium bicarbonate. Analysis of the resulting
compound revealed the presence of the corresponding keto
form, 5Me-OQE (11), resulting from tautomerization in basic
media. The ATR-IR spectrum of 5Me-OQE (11) is similar to
that of the 7Me-OQE (quinolone form) in the high-frequency
region. However, as described in detail below, matrix isolation
studies on this sample show that during sublimation 5Me-OQE
converts extensively to the most stable in the gas phase
quinoline form (5Me-HQE).
Molecular Structure of Tautomers of 5Me-HQE. The
tautomerism involving the 4-quinolone (oxoquinoline)/4-
hydroxyquinoline system may be compared to the equilibrium
between 4-pyridones (oxo form) and 4-hydroxypyridines.26 For
this system, it was found that the keto form is more stable in
the crystal and in the liquid phase or in solution, while the enol
form predominates in the vapor phase.26 For the compound in
study we may consider the equilibrium between the 4-
hydroxyquinoline and 4-quinolone (oxoquinoline) forms
shown in Figure 4: ethyl 4-hydroxy-5-methylquinoline-3-
Figure 4. Possible tautomers of ethyl 5Me-HQE considered in this
work.
carboxylate (5Me-HQE; 10), ethyl 4-oxo-5-methylquinoline-
3-carboxylate (5Me-OQE; 11), and 3-[ethoxy(hydroxy)-
methylidene]-4-oxo-5-methylquinoline (12). For simplicity,
we refer to 11 as 5Me-OQE (5-methyl-oxoquinoline-ester)
and to 12 as 5Me-OQHE (5-methyl-oxoquinoline-hydroxy
ester).
As shown above, analysis by X-ray crystallography demon-
strated the presence in the crystal of the hydrochloride salt of
the 4-hydroxyquinoline form, 5Me-HQE·HCl. It was also
demonstrated by NMR spectroscopy that the 4-hydroxyquino-
line 5Me-HQE form exists solely in solution. We also observed
that the quinolone form, 5Me-OQE, may be prepared from
alkaline washing of the salt, 5Me-HQE·HCl.
Figure 5. Conformers of the 4-hydroxyquinoline form of 5Me-HQE.
Table 1. As shown, two of the conformers (I and II) have much
lower energy than all the other forms. Forms I and II shall
represent ca. 78% and 22% of the total population in the gas
phase equilibrium at room temperature, according to
Boltzmann distribution. The higher-energy forms are exper-
imentally irrelevant under these conditions.
It is clear from the relative energies of the various conformers
of 5Me-HQE, shown in Table 1, and corresponding structures,
shown in Figure 5, that the considerably lower energy of forms
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barrier (∼30 kJ mol−1) corresponding to the highly sterically
hindered transition state with COCC dihedral equal to 0°.
Calculations at the same level of theory were also performed
on the oxoquinoline tautomers of 5Me-HQE represented in
Figure 4, specifically those implying migration of H from the
hydroxyl to the ring nitrogen (5Me-OQE, 11) or to the ester
carbonyl oxygen atom (5Me-OQHE, 12).
Table 1. Relative Energies (ΔE, kJ mol−1) and Relative
Gibbs Energies (ΔG298.15, kJ mol−1) of the Relevant
Conformers of the Tautomeric forms of 5Me-HQE
ΔE
0.0
ΔG298.15
5Me-HQE I (anti)
0.0
4.8
II (gauche)
1.9
17.3
18.3
61.3
63.0
60.6
62.3
26.6
28.7
34.4
35.4
157.8
160.2
III
17.7
20.5
57.5
59.9
57.1
59.8
23.8
27.5
31.3
33.3
151.8
154.3
The calculated conformers of 5Me-OQE and 5Me-OQHE
are represented in Figures 7 and 8. For tautomer 11, the
IV
V
VI
VII
VIII
5Me-OQE I
II
III
IV
5Me-OQHE I
II
I and II (II is higher in energy than I by only 1.9 kJ mol−1; 2.6
kJ mol−1 if zero-point corrections to energy are included) can
be correlated with the presence in these forms of the O−H···
O(carbonyl) stabilizing intramolecular H-bond interaction. Forms
III and IV are of higher energies than the most stable forms by
∼18 kJ mol−1, while all the remaining forms have relative
energies higher than 60 kJ mol−1. The intermediate relative
energies of forms III and IV can be ascribed to the presence in
these forms of the O−H···O(ester) stabilizing intramolecular H-
bond interaction. This type of H-bond is well-known to be
weaker than the O−H···O(carbonyl) one, since the carbonyl
oxygen has a better ability to act as H-bond acceptor.27,28 In
forms V to VIII, no intramolecular H-bonds exist, and repulsive
interactions between the OH and ester substituents dominate,
thus justifying their much higher energies.
Figure 7. Selected (lowest energy) forms of the 5Me-OQE tautomer.
The calculated potential energy profile for internal rotation
about the COCC dihedral, interconverting conformers I and II
is depicted in Figure 6. The two conformers are separated by a
Figure 8. Selected (lowest energy) forms of the 5Me-OQHE
tautomer. The two structures of 5Me-OQHE similar to I and II but
with the OH group pointing to the carbonyl ring oxygen atom were
found to converge barrierlessly to structures 5Me-HQE I and II,
respectively.
calculations (Table 1) show that the most stable conformer
(5Me-OQE I) is higher in energy than the most stable
conformer of the hydroxyquinoline tautomer (5Me-HQE I) by
27 kJ mol−1. For tautomer 12, the two lowest energy
conformers have larger energies than 5Me-HQE I by more
than 155 kJ mol−1 (Table 1).
The relative stability of the hydroxyquinoline and oxoquino-
line tautomers of the 7-Me substituted quinoline (5 and 6 in
Figure 2; R = CH3) was also evaluated, leading to the same
conclusions as for compound 10. In particular, the hydrox-
yquinoline form is more stable in the gas phase than the oxo
forms (the calculated energy difference between the most stable
conformers of 7Me-OQE and 7Me-HQE is ∼38.0 kJ mol−1).
Like 5Me-HQE, the 7Me-HQE hydroxyquinoline tautomer has
also two low-energy conformers of nearly the same energy (anti
and gauche in the conformation of the ethyl ester group and
Figure 6. B3LYP/6-311++G(d,p) calculated relaxed potential energy
profile for interconversion between conformers I and II of 5Me-HQE.
small energy barrier (3.5 kJ mol−1 in the I → II direction; 1.6 kJ
mol−1 in the reverse direction), the transition state (with two
symmetry equivalent forms) having the COCC dihedral equal
to ∼126°. Direct interconversion between the two symmetry-
equivalent gauche (II) forms implies crossing a large energy
F
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exhibiting an OH···OC intramolecular H-bond) that are
similar to the two most stable forms of 5Me-HQE). This allows
to conclude that, at least from the qualitative point of view,
both the tautomeric and conformational patterns in the studied
molecules are not significantly affected by the position of the
methyl substituent on the benzenic ring of the quinoline
moiety.
In order to get some clues on reasons for the calculated
relative energies of 5Me-HQE and its two investigated
tautomers (5Me-OQE, 11 and 5Me-OQHE, 12) we have
estimated the aromaticity of the two rings in the molecules,
using two well-known aromaticity indexes, the harmonic
oscillator measure of aromaticity (HOMA index), defined by
Kruszewski and Krygowsky29 and then generalized by
Krygowsky,30 and the Bird index.31
Qualitatively, the two indexes agree with each other: In 5Me-
HQE, both rings are aromatic, with the heteroaromatic ring
being only slightly less aromatic than the benzenic ring;
however, in both 5Me-OQE and 5Me-OQHE, the nitrogen-
containing ring is essentially non-aromatic, the benzenic ring
being in these cases somewhat more aromatic than in 5Me-
HQE. The reduced aromaticity of the heterocyclic ring in both
5Me-OQE and 5Me-OQHE compared to 5Me-HQE can then
also be considered a relevant factor rendering these species
more energetic than 5Me-HQE.
Matrix-Isolation FTIR and Photochemical Experi-
ments. From the theoretical results presented in the previous
section, it is likely to expect prevalence of 5Me-HQE (forms I
and II), in the gas phase. However, taking into account the low-
energy barrier for conversion of conformer II into the most
stable conformer I (only 1.6 kJ mol−1), we can expect that at
least partial conversion of II into I takes place during
preparation of the low temperature matrices prepared from
deposition of the vapor of the compound onto the cold (15 K)
IR transparent CsI optical substrate of the cryostat (conforma-
tional cooling).32 Since the gas-phase population of II is already
small (∼22%, according to the performed calculations), and the
predicted IR spectra for the two conformers are quite similar
(see the B3LYP/6-311++G(d,p) calculated IR spectra for the
two conformers in Figure S13), presence of conformer II in the
studied argon matrix could not be safely established. In fact, the
IR spectrum of 5Me-HQE isolated in solid argon can be fairly
well reproduced by the calculated spectrum of conformer I
alone (Figure 9).
The HOMA value can be obtained making use of eq 1:
n
α
n
HOMA = 1 −
(R − Ri)2
∑
opt
(1)
i=1
where n is the total number of the atoms considered, Ri a bond
length, and α and Ropt are precalculated constants given in
original paper26 for each type of atom pair (257.7 and 1.388 for
C−C and 93.52 and 1.334 for C−N, respectively). If HOMA =
1, the length of each bond is identical to optimal value Ropt, and
thus the ring is fully aromatic. If HOMA = 0, the ring is
completely non-aromatic. If HOMA assumes a significantly
negative value, then the ring shows anti-aromaticity character.
Despite its simplicity, this index has been found to be one of
the most effective structural indicators of aromaticity and a
good measure of π-electron delocalization.
The Bird index is another geometry-based quantity aimed at
measuring aromaticity. It can be obtained through eqs 2 and 3:
BI = 100[1 − (V/V )]
(2)
k
∑ (N − N)2
̅
100
N
̅
i,j
a
Ri,j
i
V =
N =
− b
i,j
n
(3)
In eq 3, i cycles all of the bonds in the ring, j denotes the
atom next to atom i, n is the total number of the bonds
considered, N denotes Gordy bond order, N is the average
value of the n values, and Ri,j is a bond length. Parameters a and
b are predefined for each type of bond, being 6.8 and 1.71 for
C−C and 6.48 and 2.00 for C−N, respectively. VK is a
predetermined reference value for V; for six-membered rings,
this value is 33.2. The closer the Bird index is to 100, the
stronger the aromaticity is.
The calculated HOMA and BI indexes for 5Me-HQE, 5Me-
OQE, and 5Me-OQHE tautomers, using the B3LYP/6-311+
+G(d,p) calculated bond lengths, are given in Table 2.
Table 2. Calculated Aromaticity Indexes (HOMA and Bird)
for 5Me-HQE Tautomers (10−12)
Figure 9. IR spectrum of the as-deposited argon matrix of 5Me-HQE
(top) and calculated spectrum for conformer I (bottom).
tautomer
10
11
12
Annealing of the matrix up to about 40 K led to small, but
perceptible changes in the relative band intensities. These
might be taken as indication that conformer II could have
partially survived to matrix deposition and is being converted
into I upon the annealing. However, such intensity changes can
also be due to matrix-site conversions or change of relative
intrinsic intensities of the different bands with temperature,
HOMA
C5N-ring
C6-ring
BI
0.73
0.76
0.04
0.90
−0.07
0.89
C5N-ring
C6-ring
78.7
80.0
57.3
88.3
60.2
88.1
G
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Table 3. Observed IR Frequencies for 5Me-HQE in Argon Matrix (15 K) and B3LYP/6-311++G(d,p) Calculated Frequencies
a
and IR Intensities for Conformers I and II
H
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Table 3. continued
a
b
Wavenumbers (ν) in cm−1; IR intensities (IIR) in km mol−1. Assignments and approximate descriptions are specific for conformer I; ν, stretching;
δ, in-plane-bending; γ, out-of-plane rocking; τ, torsion; w, wagging; tw, twisting; r1 and r2 refers to the benzene and heteroaromatic ring, respectively;
b
et, ethyl group; s, symmetric; as, antisymmetric; n.obs., non-observed; n.i., not investigated. As it is common when the molecule contains a very
strong intramolecular H-bond, the νOH band is very broad and difficult to distinguish from the baseline. Moreover, in the present case the spectral
region where it is expected to occur is quite congested due to the presence of the bands due to the CH stretching modes and also those due to the
stretching vibration of the co-deposited H−Cl (see Experimental Section). Under these circumstances, we investigated the precise assignment of this
vibration.
which, in view of the low barrier for II → I conformational
interconversion, appear as more plausible explanations for the
observations.
The proposed assignments for the bands observed in the
experimental spectrum (at 15 K) are summarized in Table 3. In
this table, the theoretical wavenumbers and IR intensities for
both conformers I and II of 5Me-HQE are also provided.
It is worth mentioning that no traces of 5Me-OQE or 5Me-
OQHE tautomers could be found in the experimental IR
spectra (see Figure S14), clearly revealing that the species
resulting from sublimation of the used hydrochloride salt
(5Me-HQE·HCl) are solely HCl and 5Me-HQE, i.e., the
tautomeric species of the compound present in the crystalline
solid did not convert, upon sublimation, to other tautomer.
Quite interestingly, the infrared spectrum of a matrix
resulting from deposition of the material obtained from 5Me-
HQE·HCl after alkaline washing (5Me-OQE, according to the
performed ATR experiments) revealed that the 5Me-OQE keto
Figure 10. Fragment of the IR spectra showing photolysis (130 min.; λ
> 220 nm) of 5Me-HQE in argon matrix. In black: IR spectrum
form undergoes extensive tautomerization to the most stable
hydroxyquinoline form (5Me-HQE) upon sublimation, since
obtained prior to irradiation; and in purple: IR spectrum obtained after
this spectrum is identical to that obtained upon deposition of
irradiation. Bands due to CO, CO2, and ketene species are identified
(see text).
the unwashed 5Me-HQE·HCl sample.
In situ broadband UV (λ > 220 nm) irradiation of matrix
isolated 5Me-HQE for 130 min led to consumption of about
one-third of the original compound. However, the number of
new bands detected in the spectra of the photolyzed matrix was
very small (Figure 10). These new bands appear at 2343 cm−1,
indicating formation of CO2,33 in the 2156−2106 cm−1 range,
and at 1734 cm−1. The bands in the 2156−2106 cm−1 range
shall be due to both CO and ketene species.34−37 The
photoproduction of ketene species from 5Me-HQE is
supported by the following facts: (i) ketenes are well-known
to give rise to a very intense (>900 km mol−1) infrared band in
the 2150−2100 cm−1 range due to the νCCO
antisymmetric stretching, which is very sensitive to the specific
conformation adopted by the ketene,37 thus in general
appearing as a band exhibiting several components, as it is
observed in the present case; (ii) formation of ketenes has been
observed for similar compounds isolated in matrices and
subjected to similar irradiation conditions, for example, methyl
p-hydroxybenzoic acid (methyl paraben) and triclosan.38,39 The
mechanism for ketene formation involves most probably the
initial formation of the 5Me-HQE phenoxyl radical, as observed
for methyl p-hydroxybenzoic acid and triclosan38,39 and for
phenol derivatives in general (in the case of phenol and phenol-
d5, the formed radical could be detected and characterized
spectroscopically in the cryogenic matrices).40 Subsequent
recombination of the hydrogen radical and ring-opening by
I
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cleavage of an α-bond to the OH ring-substituent leads to
production of the ketene; (iii) the photoproduced ketene
contains a carbonyl bond, whose absorption band in the
infrared is also expected to be intense (∼200−300 km
mol−1).38 Thus, the new band observed at 1734 cm−1 also
supports the formation of ketene species upon photolysis of
5Me-HQE in argon matrix. Note that, besides these two bands,
all other bands ascribed to ketenes are expected to be of low
intensity and, in most of the cases, to overlap with bands due to
the reactant, which explains the non-observation of additional
ketene bands in the spectra of the photolyzed matrix. Similarly,
no bands were observed, in the spectra of the photolyzed
matrix, that could be attributed to the photoproducts of the
decarboxylation and decarbonylation reactions of the ester
moiety of 5Me-HQE, accompanying the production of the
identified CO2 and CO.
In summary, the performed photochemical experiments
indicate that 5Me-HQE is relatively photostable. Upon
continuous broadband UV (λ > 220 nm) irradiation of the
matrix-isolated compound for 130 min, only about one-third of
the compound reacted, giving rise to an open-ring ketene
isomeric species. Decomposition of the compound was also
observed, resulting in CO2 and CO release.
Biological Activity and cLog P Calculations. As pointed
out in the Introduction, the knowledge of tautomeric and
conformational preferences for compounds 7Me-OQE (5, R =
CH3) and 5Me-HQE (10) is important for predicting and/or
explaining biological activity. According to docking studies
performed for selected quinolone 3-esters on yeast bc1,14 the 4-
oxoquinoline 3-ester form 7Me-OQE should present a much
better pharmacological profile as P. falciparum bc1 inhibitor than
its 4-hydroxyquinoline 3-ester isomer 5Me-HQE, with the N−
H and 4-oxo groups of 7Me-OQE being involved in important
interactions inside the enzyme pocket. However, a change in
structure will translate in change of chemical and physical
properties (like solubility), with possible impact on pharmaco-
logic performance.
We determined the clog P values for compounds prepared,
7Me-OQE and 5Me-HQE, and for corresponding tautomers,
7Me-HQE and 5Me-OQE. The antimalarial activity of 7Me-
OQE and 5Me-HQE were evaluated against the chloroquine-
resistant strain Dd2 of P. falciparum. Results are presented in
Table 4. As shown in this table, the keto forms (5 and 11) have
5-Methyl-4-hydroxyquinoline 3-ester, 5Me-HQE, exhibited
activity against P. falciparum Dd2 at micromolar concentrations
(IC50 of 5.89 μM). The oxo-tautomer, 5-methyl-4-oxoquinoline
3-ester, 5Me-OQE, is energetically less stable than 5Me-HQE
by more than 20 kJ mol−1 in the gas phase and was not
detected in solution (DMSO) or in the condensed phase
(except in the material resulting from alkaline washing of the
hydrochloride salt of the compound). It was not evaluated
against P. falciparum Dd2. However, from the viewpoint of
keto−enol tautomerism, 5Me-HQE may be compared to 7Me-
OQE, prepared by thermal cyclization in Dowtherm A
(Scheme 1; route c). The difference between 5Me-OQE and
7Me-OQE resides solely on the position of the methyl
substituent, which should not affect the keto−enol equilibrium.
4-Oxoquinoline 7Me-OQE was evaluated against P. falciparum
Dd2 showing antiplasmodial activity only slightly higher than
5Me-HQE (IC50 of 3.03 μM). These results may look
somewhat surprising if we consider published results from
docking studies, since the 4-hydroxy and 4-oxo tautomers
exhibit similar activity. However, it is worth considering the
lower value of clog P for 7Me-OQE, compared to 5Me-HQE,
so the better pharmacodynamic performance may be offset by
the poor bioavailability. This is in keeping with the proposal of
optimizing 7-substituted quinolone 3-esters 5 by tailoring the
substituent in position 7. On the other hand, understanding the
micromolar activity of 5Me-HQE requires further investiga-
tions. The 4-hydroxyquinoline 3-ester chemotype may bind
differently to the same pocket or may even switch to the Qi site
of the bc1 complex. Such a change in the primary target for the
quinolone ester class might enhance activity versus atovaquone-
resistant strains. Docking studies should be undertaken with
this class, using relevant enzyme targets.
CONCLUSIONS
■
In recent years, quinolone 3-esters were investigated as
inhibitors targeting the Qo site of the bc1 protein complex of
P. falciparum. Docking studies performed in silico at the yeast
Qo site suggested a key role for residues His182 and Glu272 (in
the target ISP) in the recognition of high potency inhibitors
and established that the 4-oxo and N−H groups appear to be
important to the inhibitory activity of quinolone 3-esters
toward P. falciparum bc1 protein complex.14
Although some 7-substituted quinolone 3-esters appear
highly promising as drug leads, with activity against both the
enzyme and the parasite at low nanomolar concentrations, the
compounds present liabilities, such as poor aqueous solubility
and low oral bioavailability. Preliminary results from SAR
studies indicated that optimization could be achieved by tuning
the nature of substituent at position 7.14 However, considering
that the information from docking studies in silico establishes a
key role for 4-oxo and the N−H groups in drug-target
interactions, the possibility of tautomerism between 4-
oxoquinoline and 4-hydroxyquinoline forms (5, 6; Figure 2)
should not be neglected. Tautomerization will translate in
alteration of chemical and physical properties, with con-
sequences for pharmacokinetic and pharmacodynamic profiles
and should therefore be investigated.
We describe the synthesis and structure of 7Me-OQE and
5Me-HQE. 7Me-OQE was obtained from 9 by thermal
cyclization in Dowtherm A at 250 °C (see Scheme 1, route
c) and isolated as sole product. 5Me-HQE was also obtained as
sole product from compound 9, by conducting the reaction in
the presence of POCl3, at 100 °C. The crystal structures of
Table 4. cLog P for 7Me-OQE (5), 7Me-HQE (6), 5Me-
HQE (10), and 5Me-OQE (11) and in Vitro Antimalarial
Activities of 5 and 10
7Me-OQE
7Me-HQE
5Me-HQE
(10)
5Me-OQE
(11)
(5)
(6)
a
clog P
SD
1.90 0.40
2.88 0.51
2.87 0.51
1.99 0.31
IC50 (μM)
Dd2 SD
3.03 0.35
n.d.
5.89 1.54
n.d.
a
By ALog PS2.1.
a lower clog P than the corresponding hydroxy forms (6 and
10). Thus, the keto forms have a higher aqueous solubility, but
their reduced lipophilicity decreases the passage through
biological membranes. So, the clog P values exhibited by the
hydroxy forms appear more adequate from a bioavailability
viewpoint, although their pharmacodynamic profile as inhib-
itors of the P. falciparum bc1 complex is expected to be less
favorable, in view of the results from docking studies.
J
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malonate derivative 9 and 5Me-HQE were investigated by X-
ray crystallography.
studies should be undertaken with this class, using relevant
enzyme targets.
5Me-HQE and 7Me-OQE were theoretically investigated
(B3LYP/6-311++G(d,p)). A detailed analysis of conforma-
tional and tautomeric preferences is provided, and the
possibility of quinolone-hydroxyquinoline equilibria is eval-
uated. Calculations show preference for the hydroxyquinoline
form in both compounds. The difference between the lowest
energy hydroxyquinoline and quinolone forms is 27 and 38 kJ
mol−1 for 5Me-HQE and 7Me-OQE, respectively. The
considerably lower energy of hydroxyquinoline forms can be
correlated with the presence in these forms of a O−H···
O(carbonyl) stabilizing intramolecular H-bond interaction.
HOMA and BI aromaticity indexes were determined for
5Me-HQE and its 5Me-OQE and 5Me-OQHE tautomers,
using the B3LYP/6-311++G(d,p) calculated bond lengths.
Qualitatively, the two indexes agree with each other. While in
5Me-HQE both rings are aromatic, with the heteroaromatic
ring being only slightly less aromatic than the benzenic ring, in
both 5Me-OQE and 5Me-OQHE the nitrogen-containing ring
is essentially non-aromatic; the benzenic ring being in these
cases somewhat more aromatic than in 5Me-HQE. The
reduced aromaticity of the heterocyclic ring in both 5Me-
OQE and 5Me-OQHE compared to 5Me-HQE can then also
be considered a relevant factor, rendering these species more
energetic than 5Me-HQE.
The structure of monomeric 5Me-HQE was also studied
using matrix isolation coupled to FTIR spectroscopy. No traces
of 5Me-OQE or 5Me-OQHE tautomers were found in the
experimental matrix isolation IR spectra, clearly revealing that
the species resulting from sublimation of the used hydro-
chloride salt (5Me-HQE·HCl) are solely HCl and 5Me-HQE,
i.e., the tautomeric species of the compound present in the
crystalline solid did not convert to other tautomer, upon
sublimation. On the contrary, when solid 5Me-OQE was used
as starting material in the matrix isolation experiments,
extensive tautomerization of the compound was observed,
resulting in the sole observation of the quinoline tautomeric
form, 5Me-HQE.
Upon continuous broadband UV (λ > 220 nm) irradiation of
the matrix-isolated 5Me-HQE for 130 min, only about one-
third of the compound reacted, giving rise to an open-ring
ketene isomeric species. Decomposition of the compound was
also observed, resulting in CO2 and CO release. Thus, 5Me-
HQE appears to be relatively photostable.
The antiplasmodial activity of 4-oxoquinoline 7Me-OQE and
4-hydroxyquinoline 5Me-HQE against chloroquine and me-
floquine-resistant P. falciparum strain Dd222 was evaluated.
Both compounds exhibited activity at low micromolar
concentrations with IC50 values of 3.03 μM for 7Me-OQE
and 5.89 μM for 5Me-HQE. The 4-oxoquinoline is only slightly
more active than the 4-hydroxyquinoline. Given the low clog P
value for 7Me-OQE compared to 5Me-HQE, a better
pharmacodynamic performance may be offset by the poor
bioavailability.
EXPERIMENTAL SECTION
■
General Methods. All reagents and solvents were purchased from
commercial sources and used as received. When required, solvents
were freshly distilled from appropriate drying agents before use. The
reactions were monitored by TLC, using silica gel F254 plates.
Purifications by flash column chromatography were performed with
40−63 mesh silica gel. When required, inorganic solids were removed
by filtration through a layer of Celite 512 medium. NMR (400 MHz)
spectra for compounds, in appropriate solvents (d6-DMSO or d-
chloroform), were measured using TMS as the internal reference (δ =
0.0 ppm). Chemical shifts (δ) are described in parts per million
(ppm). Splitting patterns are designated as s (singlet), d (doublet), t
(triplet), q (quartet), and m (multiplet). NMR spectra for target
compounds, operated at 400 and 100 MHz, for 1H and 13C,
respectively, are provided in Figures S2, S3, and S5−S9. Melting
points were recorded and are uncorrected.
Preparation of 3-Methylaniline (m-Toluidine) (8). 3-Nitro-
toluene (7) (4 mL; 33.75 mmol) was dissolved in a mixture of
methanol and water (1:1; 60 mL). Ammonium chloride (6.7 equiv)
was added, followed by iron powder (4.2 equiv), and the resulting
mixture was stirred vigorously at 60 °C for 4 h. The reaction mixture
was cooled, then diluted with methanol, and filtered through Celite.
The filtrate was concentrated by evaporation under reduced pressure,
and the aqueous residue was extracted with EtOAc. The combined
organic extracts were dried over MgSO4, filtered, and evaporated to
dryness. 3-Methylaniline (8) was recovered as brown oil (3.4 g; 94%
1
yield). H NMR (400 MHz, (CD3)2SO) δ 6.88 (t, J = 7.6 Hz, 1H),
6.34 (m, 3H), 4.88 (s, 2H), 2.14 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 147.4, 139.1, 130.2, 119.4, 115.8, 113.0, 22.3. MS m/z (GC-
TOF, EI+): 107.1490 ([M]+). These results are compatible with those
described in the literature about this compound.41
Preparation of Diethyl 2-[((3-Methylphenyl)amino)-
methylene]malonate (9). 3-Methylaniline (8) (3 mL; 28 mmol)
and diethyl ethoxymethylenemalonate (5.7 mL; 28 mmol) were stirred
at 100 °C, overnight. Purification by column chromatography (eluting
with DCM) followed by recrystallization in ethanol and DCM yielded
diethyl 2-[((3-methylphenyl)amino)methylene]malonate (9) as a
creamy crystal (7.1 g; 92%). Melting range 38−39 °C. 1H NMR
(400 MHz, (CD3)2SO) δ 10.63 (d, 1H), 8.40 (s, 1H), 7.25 (t, J = 7.1
Hz, 1H), 7.18−7.06 (m, 2H), 6.96 (s, 1H), 4.14 (m, 4H), 2.29 (s, 3H),
1.23 (m, 6H). 13C NMR (101 MHz, (CD3)2SO) δ 168.0, 165.4, 151.5,
139.7, 139.6, 129.9, 125.8, 118.3, 115.0, 93.4, 60.1, 59.9, 23.4, 14.7,
14.6. MS m/z (GC-TOF, EI+): 277.1312 ([M]+), 231.0901 ([M −
OEt]+), 203.0962 ([M − CO2Et]+), 186.0569 ([M − 2xOEt]+),
158.0745 ([M − CO2Et and OEt]+), 130.0682 ([M − 2xCO2Et]+).
CHNS for C15H19NO4 (9) requires: C 64.97, H 6.91, N 5.05; found:
C 65.07, H 6.95, N 4.77.
Preparation of 7Me-OQE (5, R = CH3). Diethyl 2-[((3-
methylphenyl)amino)methylene]malonate (9) (1.07 g; 3.9 mmol)
was suspended in Dowtherm A (10 mL), under a nitrogen
atmosphere, and the mixture was heated at 250 °C for 3 h. The
reaction mixture was cooled to room temperature. The solid
precipitate was filtered, washed with hexane and diethyl ether, and
dried to afford ethyl 4-oxo-7-methylquinoline-3-carboxylate (5) as
1
white powder (0.11 g; 12%). Melting range 282−284 °C. H NMR
(400 MHz, (CD3)2SO) δ 12.17 (s, 1H), 8.49 (s, 1H), 8.04 (d, J = 8.2
Hz, 1H), 7.38 (s, 1H), 7.24 (d, J = 8.2 Hz, 1H), 4.25−4.17 (m, 2H),
2.44 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H). 1H NMR (400 MHz, CDCl3) δ
12.20 (s, 1H), 8.52 (s, 1H), 8.26 (d, J = 8.1 Hz, 1H), 7.38 (s, 1H), 7.25
(d, J = 8.2 Hz, 1H), 4.35 (2H), 2.48 (s, 3H), 1.39 (3H). 13C NMR
(101 MHz, (CD3)2SO) δ 176.0, 165.0, 143.5, 140.6, 140.0, 131.6,
127.4, 125.3, 117.0, 111.0, 59.6, 23.4, 14.4. HRMS (CI) for
C13H14NO3 ([M + H]+) requires 232.0974; found 232.0977. CHNS
for C13H13NO3 (5, R = CH3) requires: C 67.53, H 5.67, N 6.06;
found: C 67.29, H 5.63, N 5.96.
Our results support the proposal of optimizing 7-substituted
4-oxoquinoline 3-esters 5 by tailoring the substituent in
position 7 and also expose the relevance of exploring 4-
hydroquinoline 3-esters as scaffolds. The 4-hydroxyquinoline 3-
ester chemotype may bind differently to the same pocket or
may even switch to the Qi site of the bc1 complex. Such a
change in the primary target for the quinolone ester class might
enhance activity versus atovaquone-resistant strains. Docking
K
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Preparation of 5Me-HQE (10). Diethyl 2-[((3-methylphenyl)-
amino)methylene]malonate (9) (1.37 g; 5 mmol) was suspended in
phosphoryl chloride (15 mL), under a nitrogen atmosphere, and the
resulting mixture was refluxed overnight at 97 °C. The excess of
phosphoryl chloride was evaporated under reduced pressure. The
resulting residue was cooled to 90 °C and diluted with isopropanol.
The mixture was left to cool slowly, while a solid precipitate was
formed. After filtration, washing with cold isopropanol and then with
cold light petroleum ether, a mixture of a powder and orange crystals
was recovered. The crystalline product was identified as ethyl 4-
hydroxy-5-methylquinolinium chloride 3-carboxylate (0.25 g; 22%
yield), as confirmed by X-ray crystallography. Melting range 146−147
structures were solved by direct methods using SHELXT-2014/4.48
Refinements were carried out with the SHELXL-2014/7 package.49 All
refinements were made by full-matrix least-squares on F2 with
anisotropic displacement parameters for all non-hydrogen atoms see
tables in Sections A and B in the Supporting Information for details
regarding crystallographic analysis of compound 9 and of 5Me-HQE·
HCl. In the latter compound the ethyl group is disordered over two
positions with relative occupancies of 70:30%. Appropriate SADI and
ISOR restraints were applied to the C atoms of this group. All of the
hydrogen atoms (except for those of the disordered part) could be
located on a difference Fourier synthesis; their positions were refined
as riding on parent atoms with an isotropic temperature factor using
SHELXL-2014/7 defaults that constrain these atoms to idealized
positions, except for the coordinates of the H atom of the amino group
of compound 9, involved in strong H-bonding, were freely refined.
The X-ray analysis unambiguously demonstrate the presence of diethyl
2-[((3-methylphenyl)amino)methylene]malonate (9) and 5Me-HQE·
HCl, respectively (see Figures S4 and S11, and Sections A and B in
Supporting Information).
In Vitro Antimalarial Activity and clog P Calculations.
Laboratory-adapted chloroquine and mefloquine-resistant P. falcipa-
rum Dd222 was continuously cultured as previously described by
Trager and Jensen50 and synchronized by sorbitol treatment prior to
the assays.51 Staging and parasitaemia were determined by light
microscopy of Giemsa-stained thin blood smears. Antimalarial activity
of the compounds was determined using the SYBR Green I assay as
previously described,49,50 with modifications. Briefly, early ring stage
parasites (>90% of rings, 3% hematocrit and 1% parasitaemia) were
tested in triplicate in a 96-well plate and incubated with the
compounds for 48h (37 °C, 5% CO2), parasite growth was assessed
with SYBR Green I. Each compound was tested in concentrations
ranging from 10 to 0.002 μM. Fluorescence intensity was measured
with a multimode microplate reader (Dynex Triad, Alfagene) with
excitation and emission wavelengths of 485 and 535 nm, respectively,
and analyzed by nonlinear regression using GraphPad Prism 5 demo
version to determine IC50. cLog P calculations were performed using
ALog PS2.1, as shown in http://www.vcclab.org/lab/alogps/.
1
°C. H NMR (400 MHz, (CD3)2SO) δ 8.38 (s, 1H), 7.49 (t, J = 7.1
Hz, 1H), 7.44 (d, J = 8.2 Hz, 1H), 7.10 (d, J = 8.2 Hz, 1H), 4.18 (q,
1
2H), 2.78 (s, 3H), 1.26 (t, 3H). H NMR (400 MHz, CDCl3) δ 9.26
(s, 1H), 8.51 (d, 1H), 7.91 (t, 1H), 7.59 (d, 1H), 4.58 (q, 2H), 3.01 (s,
3H), 1.50 (t, 3H). 13C NMR (101 MHz, (CD3)2SO) δ 171.1, 171.1,
161.3, 144.6, 144.1, 140.1, 140.6, 132.0, 127.9, 117.3, 114.3, 60.1, 23.9,
14.8. MS m/z (GC-TOF, EI+): 231.0899 ([M]+). NMR and MS data
are compatible with 5Me-HQE (compound 10). The powder was
submitted to elemental analysis. CHNS for C13H13NO3 (10) requires:
C 67.53, H 5.67, N 6.06; found: C 67.43, H 5.63, N 6.03.
The crystalline 5Me-HQE·HCl washed with aqueous sodium
bicarbonate and the remaining mixture was extracted with chloroform.
The organic layer was evaporated to dryness to afford an amorphous
powder that was characterized by ATR-IR and proved to be the keto
form, 5Me-OQE, showing conversion of the 4-hydroxyquinoline form
to the 4-oxoxyquinoline tautomer in basic media.
Computational Methods. All theoretical calculations were
performed using Gaussian 09,42 at the DFT(B3LYP)/6-311++G(d,p)
level of theory.43−46 Calculated harmonic vibrational wavenumbers
were uniformly scaled by 0.978. The scaled calculated frequencies,
together with the calculated intensities, were used to produce the
simulated spectra shown in the figures; bands were broadened by
Lorentzian function, with a full-width-at-half-maximum of 2 cm−1. The
potential energy profiles for internal rotation were calculated with the
driving coordinate incrementally fixed and all the other geometrical
parameters optimized.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.5b02169.
Matrix Preparation, Infrared Spectroscopy, and Irradiation
Experiments. Matrices were prepared by heating a solid sample of
the hydrochloride salt of 5Me-HQE or of 5Me-OQE (resulting from
the alkaline washing of the hydrochloride 5Me-HQE salt) in a
miniature thermoelectrically heatable glass furnace placed in the
vacuum chamber of the cryostat (APD Cryogenics closed-cycle helium
refrigerator with a DE-202A expander). Upon sublimation, the salt
released HCl, as testified by observation of the characteristic bands of
HCl isolated in an argon matrix at 2888, 2869, and 2854 cm−1
(monomer), 2856, 2818 cm−1 (dimer), 2787 cm−1 (trimer), and 2748
cm−1 (tetramer).47,44 Together with HCl, 5Me-HQE vapor was
formed, being codeposited with a large excess of argon (N60; supplied
by commercial supplier) onto the CsI substrate of the cryostat, kept at
17 K during deposition of the matrix. When the 5Me-OQE solid
sample was used to prepare the low temperature matrices, the
obtained argon matrices were shown to contain only the
hydroxyquinoline tautomeric form of the compound, testifying the
occurrence of the quinolone → quinoline tautomerization upon
sublimation.
■
S
Representative quinolones as antibacterial agents; 1H
and/or 13C NMR spectra of 8, 9, 7Me-OQE, and 5Me-
HQE; ATR-IR spectra and table of observed ATR-IR
frequencies for 5Me-HQE and 7Me-OQE crystals;
calculated infrared spectra of its two lower energy
conformers; table of experimental (X-ray, single-crystal)
details of the crystallographic studies; ORTEPII plot of
the compound 9 and 5Me-HQE·HCl (PDF)
Crystallographic data for 5Me-HQE·HCl (CIF)
Crystallographic data for 9 (CIF)
AUTHOR INFORMATION
■
The IR spectra were recorded in the range 4000−400 cm−1, with a
resolution of 0.5 cm−1, using a Thermo Nicolet 6700 Fourier-
transform infrared (FTIR) spectrometer, equipped with a KBr beam
splitter and a deuterated triglycine sulfate (DTGS) detector. UV
irradiation of the matrices was done through the outer quartz window
of the cryostat, using a HBO200 high-pressure Hg(Xe) lamp.
ATR- FTIR Experiments. The ATR-FTIR spectra were obtained
using a germanium crystal in a Nicolet iN10 MX infrared system with
a MCT-A detector and a KBr/Germanium optics. The spectral
resolution used was 2 cm−1, with aperture and number of scans of 80
and 64, respectively.
Corresponding Authors
*E-mail: rfausto@ci.uc.pt.
*E-mail: mcristi@ualg.pt.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors gratefully acknowledge Fundaca
a Tecnologia (FCT), Portugal (Project PEst-C/MAR/LA0015/
603 2013), cofounded by QREN-COMPETE-UE and CCMAR
for generous financial support. The Coimbra Chemistry Centre
■
̧
o para a Cien
̂
cia e
̃
Crystallographic Analysis. X-ray data were collected using small
single crystals of the compounds in study. The crystallographic
L
DOI: 10.1021/acs.joc.5b02169
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
(20) Nilsen, A.; LaCrue, A. N.; White, K. L.; Forquer, I. P.; Cross, R.
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Saenz, F. E.; Morrisey, J. M.; Steuten, J.; Mutka, T.; Li, Y.; Wirjanata,
G.; Ryan, E.; Duffy, S.; Kelly, J. X.; Sebayang, B. F.; Zeeman, A.-M.;
Noviyanti, R.; Sinden, R. E.; Kocken, C. H. M.; Price, R. N.; Avery, V.
(CQC) is also supported by FCT through Project PEst-OE/
QUI/UI0313/2014. P.H. acknowledges FCT for the award of a
doctoral grant (SFRH/BD/81821/2011). N.K acknowledges
FCT for the award of a postdoctoral grant (SFRH/BPD/
88372/2012). NMR spectrometers used are part of The
́
M.; Angulo-Barturen, I.; Jimenez-Díaz, M. B.; Ferrer, S.; Herreros, E.;
National NMR Facility, supported by Fundaca̧ o para a Ciencia
̂
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